Many studies have been made on the reaction of hydrolyzing a cyano group of a nitrile compound to obtain the corresponding carboxyl acids, because this is a simple and easy process for obtaining carboxylic acids.
With respect to the bioreaction of hydrolyzing only a part of the cyano groups of a polynitrile compound having a plurality of cyano groups in one molecule to obtain the corresponding cyano carboxylic acid, in particular, with respect to the process for obtaining aromatic cyano carboxylic acids by selectively hydrolyzing only a specific cyano group of an aromatic polynitrile compound, many reports have been published on the reaction utilizing the specificity to the reaction of the microorganism. For example, U.S. Pat. No. 4,629,700 discloses a process of producing cyanobenzoic acids from phthalonitriles using a Rhodococcus bacterium. Also, for example, European Patent 178,106 discloses a process for producing cyano carboxylic acids and cyano carboxylic acid amides by the selective hydrolysis of a cyano group from a polynitrile compound using four genera of gram positive bacteria including the genus Rhodococcus. 
Selective hydrolysis reactions of a cyano group in a chemical synthesis are generally not suitable for the practical use because in order to perform the reaction, a complicated procedure, such as protection of a specific cyano group, is necessary.
Bioreactions are generally admitted to have high selectivity. However, when strictly inspected, they are in many cases accompanied by production of impurities due to side reaction. For example, according to the above-described process for producing a cyano benzoic acid from a phthalonitrile using a Rhodococcus bacterium, the selectivity is not 100%, but the reaction is accompanied by from 1.0% to a few % of by-products originating in the phthalonitrile. From the standpoint of the conversion from a starting material, this may be said to be an excellent process. However, in the synthesis of medicaments or fine chemicals, the behaviors of a slight amount of by-products greatly affect the capability or safety of a substance synthesized using a starting material containing the by-products. Therefore, the above-described selectivity is not sufficiently high for the starting material in this field.
In order to elevate the purity of the products, a method of obtaining a product and thereafter further purifying it may be considered. However, for example, various by-products produced in the process of biologically producing a cyano carboxylic acid from an aromatic polynitrile are very close to each other in the physical properties such as boiling point and hydrophobicity, and complete separation thereof cannot be attained by commonly used purification methods such as distillation, extraction, and salting out.
As such, in conventional processes for producing carboxylic acids by a hydrolysis reaction of a nitrile compound using microorganisms, the selectivity of the hydrolysis reaction itself is not high and the production of by-products is not sufficiently reduced.
An alternative method for producing carboxylic acids by the hydrolysis of a nitrile compound includes enzymatic reaction methods using nitrilase, or nitrile hydratase and amidase.
The nitrilase is an enzyme which catalyzes a reaction of converting a nitrile compound into a carboxylic acid and this is useful means for obtaining a carboxylic acid useful as a raw material for medical and agrochemical preparations. Examples of the microorganisms which produces this enzyme include Fusarium solani (see Biochem. J. 167, 685–692 (1977)), Nocardia sp. (see, Int. J. Biochem., 17, 677–683 (1985)), Arthrobacter sp. (see, Appl. Environ. Microbiol., 51, 302–306 (1986)), Rhodococcus rhodochrous J1 (see, Eur. J. Biochem., 182, 349–356 (1989)), Rhodococcus rhodochrous K-22 (see, J. Bacterio., 172, 4807–4815 (1990)), Rhodococcus rhodochrous PA-34 (see, Appl. Microbiol. Biotechnol., 37, 184–190 (1992)) and Rhodococcus sp. ATCC39484 (see, Biotechnol. Appl. Biochem., 15, 283–302 sp. (1992)).
From these microorganisms, nitrilase, nitrile hydratase, or amidase is produced. In order to use these enzymes in the genetic engineering, genes of some of these enzymes have been isolated and their primary structure has been determined. With respect to the nitrilase gene, genes from Rhodococcus bacteria are disclosed, for example, in JP-A-7-99980 (the term “JP-A” as used herein denotes an unexamined Japanese patent application, first publication) and JP-A-9-28382.
In recent years, attempts have been made to utilize the capability of converting a nitrile compound these microorganisms have. Particularly, for the production of compounds having a high added value, an enzyme having excellent steric selectivity or position selectivity is required. For example, JP-A-2-84198 discloses microorganisms for use in the production of an optically active α-substituted organic acid, JP-A-4-341185 discloses microorganisms for use in the production of an optically active 2-hydroxycarboxylic acid, and EPO433117 discloses microorganisms for use in the production of optically active ketoprofen.
Among these microorganisms, the Rhodococcus sp. ATCC39484 strain has been reported to have a capacity to hydrolyze aromatic polynitrile compounds having a plurality of nitrile groups with excellent position selectivity (see, U.S. Pat. No. 556,625). The compounds having a nitrile group and a carboxyl group in the molecule, which are produced by this selective nitrile degrading enzymatic system, are very effective as a synthesis block in the production of medical or agrochemical preparations. However, the nitrilase of this microorganism is relatively low in the activity on aromatic polynitrile compounds and for utilizing this property in industry, it is an essential matter to improve the productivity of the enzyme which catalyzes the reaction. However, the nitrilase gene of this microorganism, which is indispensable in the intended modification, has not yet been elucidated.
Nitrile hydratase and amidase are enzymes which catalyze the reactions of converting a nitrile compound to an amide and an amide to a carboxylic acid, respectively. By using nitrile hydratase and amidase, amides and carboxylic acids useful as starting materials for medicines, agrochemicals, etc. can be obtained from nitrile compounds. Methods for converting nitrile compounds to corresponding amides or carboxylic acids have been developed by utilizing biocatalysts, and many microorganisms having such catalytic activity have been reported (see, JP-B-56-17918 (the term “JP-B” as used herein means an examined Japanese patent application, second publication), JP-B-59-037951, JP-B-61-162193, JP-B-61-021519, JP-B-64-086889, JP-B-4-197189, JP-B-2-000470, EP0444640, etc.).
From these microorganisms, nitrile hydratase and amidase or nitrilase have been purified, and further, in order to utilize these genes in genetic engineering, the genes have been isolated and their primary structures have been determined. With respect to the nitrile hydratase gene, for example, genes derived from Rhodococcus bacteria are disclosed in U.S. Pat. No. 2,840,253 and EP0445646 (JP-A-40211379), genes derived from Pseudomonas bacteria are disclosed in JP-A-30251184, genes derived from Rhizobium bacteria are disclosed in JP-A-6-025296 and JP-A-6-303971. Also, with respect to the amidase gene, for example, genes derived from Brevibacterium bacteria and genes derived from Rhodococcus are disclosed in EP0433117. Further, genes derived from Rhodococcus erythropolis are reported in Eur. J. Biochem. 217(1), 327–336 (1993) and genes derived from Pseudomonas bacteria are reported in FEBS Lett. 367, 275–279 (1995).
Further, an invention relating to a recombinant plasmid containing both a nitrile hydratase gene and an amidase gene derived from Rhodococcus bacteria is disclosed in JP-A-5-068566.
In recent years, attempts have been made to utilize the capacity to convert a nitrile compound that these microorganisms have. Particularly, for the production of compounds having a high added value, an enzyme having excellent steric selectivity or position selectivity is required. For example, JP-A-2-84198 discloses microorganisms for use in the production of an optically active α-substituted organic acid, JP-A-4-341185 discloses microorganisms for use in the production of an optically active 2-hydroxycarboxylic acid, and EPO433117 discloses microorganisms for use in the production of optically active ketoprofen.
Among these microorganisms, the Rhodococcus sp. ATCC39484 strain has been reported to have a capacity to hydrolyze aromatic polynitrile compounds having a plurality of nitrile groups with excellent position selectivity (see, U.S. Pat. No. 556,625). The compounds having a cyano group and an amide group in the molecule or those compounds having a cyano group and a carboxyl group in the molecule, which are produced by this selective nitrile degrading enzymatic system, are very effective as a synthesis block in the production of medical or agrochemical preparations. However, the nitrilase of this microorganism is relatively low in the activity on aromatic polynitrile compounds and for utilizing this property in industry, it is an essential matter to improve the productivity of the enzyme which catalyzes the reaction. However, the related enzyme genes of this microorganism, which are indispensable in the intended modification, have not yet been elucidated for either nitrile hydratase and amidase.